EVERYTHING ON ICE! ! !
1. Thaw the RIPA buffer and protease/phosphatase inhibitors.
2. Add protease/phosphatase inhibitors into RIPA buffer (only add day-of use)
3. Transfer tissue to 1.5ml tube.
4. Add 300 µl of lysis buffer with protease/phosphatase inhibitors.
5. Homogenize with a homogenizer.
6. Sonicate the samples.
7. Add any additional lysis buffer.
8. Rotate samples for 30min at 4°C.
9. Set the centrifuge at 4°C.
10. Spin 12000rpm 10 min.
11. Transfer supernatant into new tubes.
12. Perform protein concentration assay for quantitation.
13. Store the rest of the samples in -20°C freezer for Western Blotting.
